Fluorescent measurements are useful for a variety of applications. One big area of application for such measurements is in diagnostics. However, in the making and using devices that are based on fluorescent measurements, several problems are encountered.
The use of fluorescent measurements in different applications is known in the art. For example, in patent WO 0050872(A2) and WO 9909455(A1), an apparatus capable of measuring quantities of biological or other types of samples that have been labeled using any of a variety of techniques including fluorescence, radioisotopes, enzyme activated light emitting chemicals and enzyme activated fluorescent materials is provided. The provided scanner includes a source module that preferably contains an internal laser emitting two different wavelengths of approximately the same intensity. An optional external light source may be coupled to the source module, thus adding further flexibility through the addition of other wavelengths (e.g. UV, visible, mid-IR, and IR). In NL 9000622(A), the illumination system uses a coherent light source, e.g. an argon ion laser and a microscope lens assembly where the detection system which also uses the microscope lens, can detect several fluorescing colourings at various wavelengths, as well as scattered light signals. The optical focussing and tracking system uses a contactless infra-red light technique. A confocal laser scanning microscope is used for forming an image. The invention mentioned in FR2924805 has a converter of linear polarization in radial polarization of the light beams emitted by the light source which is laid out between the light source and the interferometer thus resulting in a high resolution image. Abstract of GB 2443715 (A) describes a portable spectrophotometer suitable for harsh environments is used to identify and quantify a substance in a sample. It comprises a housing containing a light source, a probe for transmitting light from the light source to a sample to be analyzed and a probe for receiving light from the sample to be analyzed. A microprocessor comprises a reference library and an algorithm to identify a compound or class of compounds in the sample. However, the systems, methods and apparatus will allow for only one type of detection: either bulk or event based detection.
In case of identifying and enumerating specified blood cell subclasses, U.S. Pat. No. 4,284,412, states that a blood sample is first incubated with a reagent including antibodies to the lymphocyte subclass to be identified, the antibodies being directly or indirectly made fluorescently responsive to particular light (e.g. argon ion laser). The sample is illuminated, a cell at a time, by such focused coherent light, and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify and enumerate cells of the specified subclass. WO 9508118 (A1) discloses a method and apparatus for a biological sample assay comprising forming a potentially reactive system of the sample and a fluorophore-conjugated reagent specific to a target compound, separating reacted and unreacted reagent and detecting fluorophore fluorescence in one of the separated components, the detected fluorescence being at near infra-red wavelength. However, the methods involved herein are quite cumbersome and require multiple steps, and the devices are not conducive for use in harsh environments.
WO 2006118420(A1) and US 2003133840(A1) are directed towards providing convenient and economical alternatives for detection of signals arising out of an analysis device. However, they don't address the problems related to sample preparation and the analysis. Further, the sophisticated devices described in the aforementioned patent publications are not very conducive for rapid calibration and problem-identification and if necessary subsequent servicing.
An apparatus for optical testing of samples is disclosed in EP 0515129. The apparatus includes an apparatus for receiving a plurality of samples to be tested, light detection apparatus, apparatus defining light paths-extending from the plurality of samples to the light detection apparatus and apparatus for exposing the light detection apparatus to light received from individual ones of the plurality of samples along the light paths. JP 7229828 describes an invention to measure in-plane and vertical double refraction of an optical disc substrate accurately, quickly and conveniently by setting the incident plane in a plurality of directions and making an optical beam incident on the optical disc substrate. JP 2000081387(A) also describes a similar method. The invention in JP 7280741(A) detects the degree of the position shift of a true center of a wafer and a rotary center and correct the coordinates values of a foreign substance data in a surface inspecting apparatus so that at the time of observation of the foreign substance by a SEM (scanning electron microscope), a desired foreign substance can be captured easily. Similarly in JP 2001242082 (A), the sample chip containing multiple biological samples is optically scanned by means of the biological sample optical scanning device for identifying a biological sample labeled with the fluorescent material. Fluorescence from the fluorescent material excited by the light radiated from an objective lens is received by means of a light receiving member via the hollow part of a rotor to output an electric signal thus providing a biological sample optical scanning device greatly shortening an optical scanning time on a sample chip for efficient analysis and having excellent fluorescence detection sensitiveness of fluorescent material used for labeling the biological sample. DE 4307042(A1) describes the use of a laser diode which emits light in the red or near infrared wavelength region for excitation. It is possible, in combination with dye molecules whose absorption range overlaps with the laser wavelength, to reduce the background fluorescence, considerably to reduce the constructional cost, and simultaneously to use the evanescent wave when a time-saving one-step test is constructed for the optical detection of molecules, biomolecules and microorganisms.
In EP 0681178 (A1), an apparatus and method of the invention disclose a scanning imaging cytometer wherein an unprocessed biological fluid sample is reacted with a fluorescently-labeled binding agent. A spatial filter of a sufficient pinhole diameter is selected to allow simultaneous volumetric detection of all fluorescent targets in each columnar region. JP 2007020557 (A) provides an apparatus for measuring microorganisms subjected to fluorescent dyeing; wherein at a point during the measurement, if a preset numerical value is exceeded, an alarm can be given or the measurement can be suspended. WO 9835223(A1) describes a method for increasing the accuracy and the types of data measurements of laser scanned dye stained cells, in a single sample, by means of multiple assays, utilizing cell positions as a factor in merging data measurements. Change in lasers, use of different cell dye stains and different treating reagents provide additional data regarding cells of the sample and fixing of cell positions in the first assay permits merging of the data obtained in subsequent assays. Such analysis systems may be used for a single-type of analysis, however, in many practical situations, multiple analyses are required for effective identification and quantification of analytes.
EP 1219950 (A1), EP 1219951(A1) and JP 2002323437(A) discloses a method wherein the volume of single red blood cells or other particles suspended in liquids are determined by fluorescent labeling the sample. The cell volume is determined using fluorescence intensity values measured (i) in a first area comprising a single cell, (ii) in a second area close to that cell, and (iii) in said second area, after changing the cuvette thickness by a known amount. As already noted, making parts that are well-machined having no surface irregularities are difficult, especially for those parts that are generally used once, such as sample carriers. WO 8400817(A1) mentions a method and apparatus for fluorescent immunoassay which utilizes total internal reflection at the interface between a solid phase and a liquid phase of lower index of refraction to produce an evanescent wave in the liquid phase. In WO 9702482(A1), apheresis samples are incubated with a surfactant which allows the intercalating dye to enter the WBC. A scanning instrument scans, identifies and enumerates the WBC in the apheresis sample. The system uses an adaptive intensity threshold to identify target fluorescent particles. In these cases, however, sample preparation methods and reagents may be expensive, and since liquid samples are being used, it may not be conducive for transportation and handling in a remote, scant-resource, harsh environments.
A microscale binding assay, analyte binding array, and kits are disclosed in WO 9954736(A1) which exploit the mass action law to harvest analyte from a liquid sample. This approach, coupled with direct fluorescence detection in the NIR, yields maximal signal intensity and low background for optimal sensitivity. US 2007207513 (A1) also provides methods, products and kits for identifying an analyte in a sample but the method includes combining the sample with a first reactant capable of specifically coupling to the analyte. The first reactant is then coupled to beads. The method further includes identifying the analyte in the sample by detecting the modified substrate bound to the surface of the beads and/or the reactants bound to the beads. U.S. Pat. Nos. 7,300,800 and 6,838,289 utilized a combination of fluorescent labels for labeling particles and an analyte specific fluorescent analyte detection dye. The particles contain a combination of fluorescent labels for coding the particles and an analyte specific fluorescent dye. Near infrared (NIR) fluorescent labels useful in the detection system are also provided. U.S. Pat. No. 6,905,885 describe a portable pathogen detection system that accomplishes on-site multiplex detection of targets in biological samples. The system includes: microbead specific reagents, incubation/mixing chambers, a disposable microbead capture substrate, and an optical measurement and decoding arrangement. U.S. Pat. No. 6,905,881 provides a microbead-based test plates and test methods for adjusting fluorescence imaging systems involving using a plate with fluorescent microbeads bound to a surface. U.S. Pat. No. 5,747,349 provide a method and apparatus for rapid measurement of a fluid bulk analyte, requiring only microscale volumes. Several fluid bulk analytes can be measured simultaneously and, for biological samples, the cell content can also be measured simultaneously. The invention comprises reporter beads for chemical analysis of fluid bulk properties such as pH, oxygen saturation and ion content. Despite the availability of several elegant solutions, such methods and devices are useful for single type of analysis only.
U.S. Pat. No. 5,866,433 describes an optochemical fluorescence sensor with a biorecognitive layer for measuring the concentration of one or more analytes in a sample is provided with at least one island layer which is applied on a sensor substrate. The invention in U.S. Pat. No. 5,786,219 describes novel fluorescently labeled microspheres, where the microspheres possess at least one internal fluorescent spherical zone. The invention also describes the method of preparing the novel microspheres, the method of calibrating microscopy instrumentation using the novel microspheres, the method of using the novel microspheres as distinct labels for combinatorial analysis and the use of the labeled microspheres as tagging agents and tracers. U.S. Pat. Nos. 5,194,300 and 5,132,242 describe methods of making highly fluorescent latex microsphere having a diameter of less than five hundred angstroms and have more than five thousand fluorescent markers per sphere. The microspheres are prepared by reacting an acrylic latex bead with a diamine and a fluorescent amine at elevated pH. U.S. Pat. No. 5,147,609 describe an assay element suitable for use in an automated analytical test instrument for assaying a fluid sample. The element includes a thin porous member possessing a high degree of capillarity such as a fibrous mesh pad supported within a guide defined by surfaces contiguous the porous member. U.S. Pat. No. 5,104,813 provides a dilution and mixing cartridge that allows single (or multiple) dilutions of a sample with a diluent in a disposable cartridge in which a measurement, such as optical density, is made. Addition of sample to the device automatically measures the sample, and addition of diluent automatically causes a fixed ratio of sample and diluent to enter a receiving chamber, in which mixing and measurement can take place. U.S. Pat. No. 5,053,197 describes a diagnostic assay module for analytical procedures in which an optical signal developed by interaction between a component in a sample fluid, such as an analyte in a biological fluid, and one or more reagents in a resilient assay element is read by optical means. U.S. Pat. No. 4,144,452 describes a fluorometric system to determine the kind and amount of substances derived from a biological fluid (e.g., serum or urine) or tissue in which the substances to be detected (e.g., antigen, antibody, hormone or enzyme) are coated onto a substrate surface in fluorescent form. Multiple coating areas of different samples may be employed. The fluorometric system includes a source of filtered light to excite fluorescence, an optical system for conducting the excitation light to such coating, and optical systems for receiving emitted fluorescence and for detecting the same. The invention in US 2006073611(A1) relates to methods of assaying the levels of proteins or antibodies in a test sample, and in particular, it relates to a method of determining the relative abundance of a plurality of proteins in a test sample compared to a reference. U.S. Pat. No. 7,295,316 illustrates a fluorometry device and method adapted to determine concentration of spectrally distinguishable species in a biological sample with a plurality of movable optical devices. U.S. Pat. No. 7,024,061 portrays an optical scanning device for scanning with a radiation beam a substantially circular track of an information layer. U.S. Pat. No. 6,979,830 describes methods and instrumentation for performing charge coupled device (CCD)-based confocal spectroscopy with a laser spot array are provided. The methods and instruments of the invention are useful in any spectroscopic application, including, but not limited to, microscopy and microvolume laser scanning cytometry (MLSC). In U.S. Pat. No. 6,514,770 immunoassay methods for measuring the concentration of an analyte in a test specimen are described. The methods use an immunoreagent, where one of the analyte and the immunoreagent is an antigen, and the other of the analyte and the immunoreagent is an antibody which specifically binds to the antigen. U.S. Pat. No. 4,461,973 describes a method and apparatus for measuring the concentration of a substance capable of absorbing infrared, visible or ultraviolet radiation energy, the substance being in a mixture. The method and apparatus involve passing a beam of radiant energy having predetermined spectral response characteristics through the mixture, modulating the beam at a predetermined frequency. In U.S. Pat. No. 7,102,737 a method and apparatus for detection of a particular material, such as photo-resist material, on a sample surface are disclosed. A narrow beam of light is projected onto the sample surface and the fluoresced and/or reflected light intensity at a particular wavelength band is measured by a light detector. U.S. Pat. No. 6,228,652 mentions a blood analyzing instrument includes a single transducer for simultaneously measuring the DC volume, RF conductivity, light scattering and fluorescence characteristics of blood cells passing through a cell-interrogation zone. In U.S. Pat. No. 5,939,326 a device for analyzing a whole blood sample is provided. The device comprises a conventional hematology analyzer integrated with a fluorescence cyometry analyzer. U.S. Pat. No. 5,784,152 describes a method and apparatus of analyzing samples contained in a microplate. The instrument is capable of measuring fluorescence, luminescence, and/or absorption within multiple locations within a sample well.
All of the methods and devices mentioned herein suffer from the drawbacks that include at least one of being expensive, using expensive reagents and consumable/disposable parts, not capable of being used in a harsh and resource-scant environment, and are at best capable of very limited analysis, to name a few problems. Hence, there is a dire need to make available a device that can address all these drawbacks, and accordingly a method that can be adaptable to be used in such a device.